First published online 23 May 2007
doi: 10.1242/dev.02863
Development 134, 2415-2424 (2007)
Published by The Company of Biologists 2007
Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae
Yury Goltsev1,
Naoyuki Fuse2,
Manfred Frasch3,
Robert P. Zinzen1,
Gregory Lanzaro4 and
Mike Levine1,*
1 Department MCB, Division of GGD, Center for Integrative Genomics, University
of California, Berkeley, CA 94720, USA.
2 Department of Developmental Genetics, National Institute of Genetics, 1111
Yata, Mishima 411-8540, Japan.
3 Brookdale Department of Molecular, Cell and Developmental Biology, Box 1020,
Mount Sinai School of Medicine, New York, NY 10029, USA.
4 Department of Entomology, University of California, Davis, CA 95616,
USA.
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Fig. 1. Gastrulation morphology in D. melanogaster and A.
gambiae embryos. (A) Scanning electron micrographs of D.
melanogaster and A. gambiae embryos, ventral views. The apical
surface of a lateral neuroectoderm cell is colored blue and a mesoderm cell is
colored red. Note the constriction of the apical cell surface during
gastrulation. (B) Cross sections of gastrulating mosquito embryos after
hybridization with a digoxigenin-labeled twist antisense RNA probe.
The different panels show progressive time points starting with the stage
immediately preceding gastrulation (a) and ending with the completion of
gastrulation (f). Ingression of the mesoderm is best seen in e when some of
the mesoderm progenitors are inside the blastocoel while the rest are on the
surface. Staining in both top and bottom regions of e and f is due to germband
elongation.
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Fig. 2. Expression of mesoderm and neurogenic patterning genes in D.
melanogaster and A. gambiae embryos. Gastrulating embryos
(A. gambiae, left and D. melanogaster, right) were
hybridized with different digoxigenin-labeled antisense RNA probes.
(A-F) Ventral views; (G-J) lateral views. (A,B) twist
(twi); (C,D) snail (sna); (E,F)
single-minded (sim); (G,H) brinker (brk);
(I,J) intermediate neuroblasts defective (ind).
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Fig. 3. Extra-embryonic membranes. Staged A. gambiae embryos were
analyzed by scanning electron microscopy (A,B) and DIC
microscopy (D,E,F). (C) Lateral view of D.
melanogaster embryo at the end of germband extension stage; the
amnioserosa is indicated by the white arrow. The embryos in the left panels
are at the same relative stages as those shown in the right panels. The same
embryo is shown in E and F, and was colored in F to better visualize the
separate amnion (red) and serosa layers (blue). The embryos in D-F were
hybridized with an eve RNA probe. Blue arrows indicate the serosa,
red arrows indicate the amnion.
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Fig. 4. Expression of dorsal patterning genes in A. gambiae.
Embryos (A. gambiae, left and D. melanogaster, right) were
hybridized with the indicated antisense RNA probes. A-D,F are dorsal views;
E,G,H, are lateral views. (A,B) hindsight
(hnt); (C,D) tailup (tup);
(E,F) Dorsocross1 (Doc1) before germband
elongation; (G,H) Doc1 after germband elongation. The
area outlined by the red oval in A is the prospective serosa; the white arrow
in G indicates the amnion.
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Fig. 5. Expression of Dpp signaling components in A. gambiae.
Embryos (A. gambiae, left and D. melanogaster, right; all
lateral views) were hybridized with the indicated antisense RNA probes.
(A,B) decapentaplegic (dpp);
(C,D) thick veins (tkv); (E-H)
zerknullt (zen). All embryos are at the cellular blastoderm
stage, except E, which is a precellular mosquito embryo.
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Fig. 6. Expression of putative repressors in the A. gambiae serosa.
Embryos (A. gambiae left and D. melanogaster right; all
lateral views) were hybridized with the indicated antisense RNA probes.
Embryos are oriented to show lateral views. (A,B) empty
spiracles (ems); (C,D) tramtrack
(ttk).
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Fig. 7. Expansion of Dpp signaling in A. gambiae dorsal ectoderm.
Mosquito (A.g.) and fruitfly (D.m.) embryos were hybridized
with short gastrulation (sog; A,B,F) or
tolloid (tld; C,G,H) antisense RNA
probes. A,B,C, and G are lateral views; F and H are ventral views. Mosquito
(D,E) and fruitfly (I,J) embryos were also stained
with an antibody that recognizes the active, phosphorylated form of Mad, pMad.
(D,I) Lateral views; (E,J) dorsal views. The resulting staining patterns
indicate a much broader domain of Dpp signaling in the dorsal ectoderm of
A. gambiae embryos as compared with D. melanogaster.
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Fig. 8. The A. gambiae sog enhancer directs restricted expression in
transgenic D. melanogaster embryos. (C) The Cluster-Draw
program (Zinzen et al., 2006 )
identified several potential Dorsal binding clusters in the A. gambiae
sog locus. (D) The best cluster is located at the 3' end of
intron 1 (top schematic). Different genomic DNA fragments (numbered pink bars)
were attached to a lacZ reporter gene, inserted into the D.
melanogaster genome (lower schematic) and expressed in transgenic
embryos. Only fragments 1 and 2 exhibited any activity as measured by in situ
hybridization with a lacZ antisense RNA probe. (A,B)
Lateral and ventral views, respectively, of embryos after staining with the
larger fragment (the fragment 1-lacZ fusion gene).
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Fig. 9. Model for the patterning of the A. gambiae dorsal ectoderm.
(A) The diagrams represent cross-sections of early mosquito (bottom)
and fruit fly (top) embryos. The sog expression pattern (blue) is
restricted to ventral regions of mosquitoes, but is broadly distributed in
lateral regions of D. melanogaster. The ventralization of the
sog pattern in A. gambiae might cause the indicated
expansion of Dpp signaling and pMad expression (red). There is sequential
expression of Sog and Tld in both the fruitfly and mosquito embryo. However,
the two patterns are shifted towards ventral regions in the mosquito embryo.
(B) Quality of Dorsal binding sites in the Anopheles sog
enhancer (average score of 6.7) as compared with those in the
Drosophila enhancer (average score of 10.1). The score range covered
by the box contains 50% of all data points (the second and third quartiles of
distribution). The bottom and top marks correspond to maximal and minimal
score values, respectively (see Papatsenko
and Levine, 2005). The Roman numerals beneath the plots indicate
each of the three major patterning thresholds. For example, the htl
and sna enhancers are type 1 enhancers that are activated only by
high levels of the Dorsal gradient. (C) Tld is responsible for
generating a peak of Dpp signaling at the dorsal midline, resulting in a spike
of pMad activity in the Drosophila dorsal ectoderm (left panels). By
contrast, the altered patterns of tld and sog expression in
A. gambiae embryos are expected to generate two peaks of Dpp
signaling activity, resulting in the broad plateau of pMad staining in the
dorsal ectoderm (right panels). The subdivision of the dorsal ectoderm into
distinct amnion and serosa lineages can be explained on the basis of the
expanded pMad staining pattern, and the recruitment of the repressor Ttk into
the Dpp signaling network. The asterisks indicate specific regulatory linkages
that are lost in D. melanogaster. Only one of these linkages is
required for the expression of ttk, or some other serosa-specific
repressor in A. gambiae.
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© The Company of Biologists Ltd 2007
